The goal of this study is to identify underlying mechanisms, which hinder routine high-level expression of integral membrane proteins using the baculovirus expression system. In this study a rapid cloning system will be employed to generate recombinant baculoviruses expressing fluorescent-protein-tagged model membrane proteins as tools for identifying possible causes for poor expression and evaluating the effectiveness of changes in the expression system made to alleviate those bottlenecks. The fluorescent-tag will facilitate analysis of protein expression, expression kinetics, and localization by fluorescent microscopy, flow cytometry, and in gel fluorescence. Microarray analyses and RNA-silencing will be used to identify candidate host and virus genes that either impede or enhance membrane protein expression. These data will be analyzed to deduce what cellular pathways and processes may be affected. Based on these analyses, modifications in host cell and virus gene expression will be tested as means for improving membrane protein production and quality. Biochemical and molecular techniques will be used in conjunction with fluorescent protein monitoring to assess the effects of these changes on production of membrane proteins suitable for structural studies. Results from these studies will be used to design a new generation of baculovirus expression vectors and develop host cells optimized for membrane protein synthesis. PUBLIC HEALTH RELEVANCE: The human health relevance of this project is the development methods to produce membrane proteins for structural studies in order to better understand the molecular mechanisms responsible for conditions such as hypertension, cancer, Alzheimer's disease, and chronic pain that arise when membrane proteins are mutated, misfolded, or misexpressed and to aid in the design of effective and safer drugs for these conditions.